Formation and development of primordial follicles are obligatory events for successful folliculogenesis and fertility. Defects in these steps lead to ovarian dysgenesis or premature ovarian failure and ultimately infertility. Our long-term goal is to elucidate the regulatory mechanisms controlling the formation and development of the primordial follicles as a necessary prerequisite to the development of improved therapeutic management of human infertility and contraception. Although evidence suggests that growth differentiation factor 9 (GDF9) or E2 stimulates preantral follicle development, virtually nothing is known about their role in primordial folliculogenesis or the mechanisms therein. This renewal application is focused on filling this knowledge gap. During the last funded period, we have shown that inactivation of FSH during fetal development disrupts primordial folliculogenesis, which correlates with low expression of GDF9 and estrogen receptors (ESR). Further, FSH treatment upregulates GDF9 and ESR expression. Our preliminary results indicate that (1) suppression of endogenous GDF9 expression in vitro retards primordial folliculogenesis, (2) E2 stimulates primordial follicle formation in vitro, (3) fetal exposure to an FSHantiserum blocks the expression of the GDF9 receptors, such as bone morphogenetic protein receptor II [BMPRII] and transforming growth factor B receptor type I [TBRI], and blocks the expression of ESR1 and ESR2, and (4) siRNA knockdown of a G-protein coupled receptor 30 (GPR30), a transmembrane ESR in vitro attenuates the formation of primordial follicles. Based on these observations we hypothesize that GDF9 and E2 promote the differentiation of somatic cells into granulosa cells leading to the formation of primordial follicles by mechanisms that involve a concerted action of the GDF9 receptors (BMPRII and TBRI), and the ESR (ESR1, ESR2 and GPR30). We further postulate that FSH regulates the action of GDF9 and E2 by modulating the expression of their receptors. The hypothesis will be tested with the following specific aims: 1. To reveal the physiological importance of GDF9 expression in the differentiation of somatic cells into pregranulosa cells leading to primordial follicle formation. We will determine the need for intraovarian GDF9, and its mechanisms of action. 2. To examine the physiological importance of the ESR in the differentiation of somatic cells into pregranulosa cells leading to primordial follicle formation. We will determine the need for the classic ESR and GPR30 with respect to GDF9 action. We will use fetal and neonatal hamster ovaries in vivo and in vitro (primordial follicles do not appear until 8th day of life), RNAi, antisense oligodeoxynucleotides, pharmacological, morphological and molecular approaches to address the specific aims. The successful completion of this project should advance our understanding of the mechanisms involved in the morphogenesis of primordial follicles. Lay summary: The primordial follicle stock represents a nonrenewable follicular reserve for the entire reproductive life of the mammalian species, including the human, and determines the fertility and fecundity of the species. The purpose of the proposed research is to elucidate the regulatory mechanisms controlling the formation and development of the primordial follicles. The outcomes will have a significant impact on the development of improved or novel therapeutic management of human infertility and contraception.